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The Enzymatic Post-Transfer Editing Mechanism of E. coli ThreonyltRNA Synthetase (ThrRS): An MD and QM/MM Investigation Mohamed M. Aboelnga, John J. Hayward, and James W. Gauld ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b01554 • Publication Date (Web): 26 Jun 2017 Downloaded from http://pubs.acs.org on June 26, 2017

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The Enzymatic Post-Transfer Editing Mechanism of E. coli Threonyl-tRNA Synthetase (ThrRS): An MD and QM/MM Investigation. Mohamed M. Aboelnga,1,2 John J. Hayward,1 and James W. Gauld*

1

Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, N9B

3P4, Canada. 2

Department of Chemistry, Faculty of Science, University of Damietta, New Damietta, Damietta

Governorate 34511, Egypt.

ACS Paragon Plus Environment

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ABSTRACT: Threonyl-tRNA synthetase (ThrRS) is a class II aminoacyl-tRNA synthetase (aaRS), which are a ubiquitous family of enzymes that have a vital role in protein biosynthesis. In particular, it catalyses the activation and subsequent aminoacylation of its corresponding tRNAThr. Because of the close structural and electronic similarity between its cognate substrate threonine and the noncognate serine, the catalytic aminoacylation site of ThrRS is not able to fully discriminate between them. In this study we have explored multiple possible post-transfer editing mechanisms for ThrRS from Escherichia coli. The editing site is known to contain two conserved histidyl's (His73 and His186) and a cysteinyl (Cys182), all of which could act as the required mechanistic base. We have performed detailed molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) studies in which the protonation states of each of these residues was varied. Furthermore, using the various substrate-bound active site models obtained, we have examined previously proposed and alternative possible mechanisms for deaminoacylation of SertRNAThr by ThrRS in which His73 or Cys182 act as the base; 11 mechanisms in total. The present results suggest that the most feasible mechanism is obtained when both His73 and His186 are neutral, while the thiol of Cys182 is deprotonated and acts as a base. The resulting mechanism is found to occur in two steps. First, deprotonation of an active site water by the thiolate of Cys182 with its concomitant nucleophilic attack at the substrate's Ccarb center occurs with a calculated free energy barrier of 9.9 kcal/mol. The subsequent, and overall rate-limiting step, is a water-meditated proton transfer from Lys156 onto the

Ado763′-oxygen

resulting in

simultaneous cleavage of the Ado763′O—Ccarb bond with a free energy barrier of 20.8 kcal/mol. Keywords: QM/MM, Molecular Dynamics, Aminoacyl-tRNA Synthetases, Post-Transfer Editing, Hydrolysis, Cysteinyl Base, Computational Enzymology.


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INTRODUCTION: The remarkable accuracy in the translation of the genetic code of all organisms into proteins is closely tied to the outstanding specificity of the ubiquitous aminoacyl-tRNA synthetase (aaRS) family of enzymes.1,2 These enzymes are responsible for catalyzing the activation of their cognate amino acid via its reaction with ATP to form aminoacyl-AMP. Subsequently, within the same synthetic (aminoacylation) site they catalyze transfer of the activated aminoacyl group onto its corresponding tRNAaa, yielding the desired aminoacyl-tRNAaa.3 Importantly, they have been stated to be able to perform this overall process with a fidelity of 10−4, i.e. 1 aminoacylation error per 10000 reactions.2 Consequently, this crucial class of enzymes has been the subject of many experimental and theoretical studies seeking to gain deeper insight into their catalytic accuracy.2, 4 On the basis of size or chemical discrimination the active size pocket of aaRSs typically accommodate the corresponding cognate substrate amino acid while excluding noncognate amino acids.5 However, due to electronic and/or structural similarities between some amino acids (e.g. threonine, serine, and valine), it is challenging for almost half of the aaRSs to correctly distinguish between cognate and noncognate amino acids.6-7 Except for specific cases where moderate levels of mistranslation errors can be tolerated, or even necessary,8-9 reduced accuracy of translation can lead to protein misfolding, which is responsible for many fatal diseases.10 To ensure accurate translation most aaRS possess proofreading mechanisms. In the synthetic site they may use pre-transfer editing mechanisms of which the most common is hydrolysis.11 For instance, a water nucleophilically cleaves the labile phosphoester bond of a misactivated amino acid. This type of editing is observed in many enzymes; e.g. LeuRS hydrolyzes misactivated ValAMP12 and ThrRS hydrolyzes misactivated Ser-AMP.13 However, many aaRS may also exploit a distinct second editing active site – the editing site – which catalyzes post-transfer editing of misacylated tRNA.11 For example, AlaRS,14 ThrRS,15 and LeuRS16 enzymes utilize this type of correcting pathway to reject mischarged Gly-tRNAAla, Ser-tRNAThr, and Ile-tRNALeu, respectively. It has also been noted that some enzymes have a second, free-standing editing domain; for example, AlaRS has the AlaXp active site to correct any mislinked serine.17


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While it is thought that yeast mitochondrial ThrRS relies only upon pre-transfer editing,18 the ThrRSs found in all other species are suggested to also have an editing site.15 This latter site is remote from the synthetic site and is responsible for the hydrolysis of misacylated Ser-tRNAThr. Structurally, the editing domain has been observed to be species specific, with the two main types being bacterial and eukaryotic, and archaeal, and they are thought to use two different editing mechanisms.19 Our group has a long-standing interest in the mechanisms operating in aaRS enzymes, both regarding their synthetic activity and the pre-transfer editing functions of the synthetic site.20-21 The principal aim of this study is to expand the understanding of the posttransfer editing functions of threonyl-tRNA synthetase (ThrRS) enzymes. In archaeal ThrRS, for example from Pyrococcus abyssi, the editing region is located in the N-terminal domain and the editing mechanism proceeds by a universal tRNA-mediated hydrolysis mechanism.5,22,23 In contrast, in bacterial ThrRS, for instance from Escherichia coli, the editing region has a quite distinct structure and different hydrolytic editing mechanisms have been proposed. Unfortunately, despite experimental studies on various possible editing pathways, the exact mechanism remains an open question. Based on mutational and kinetic analyses it has been suggested that the binding domain’s His73, Lys156, His186 and Cys182 residues are mechanistically most important.15,24 Notably, the His73Ala and Cys182Ala mutations were observed to cause the most significant decreases in the rate of hydrolytic editing.24,25 In addition, based on X-ray crystal structures it was concluded that His73 is within hydrogen bonding distance of a water (W1) thought to be positioned near the substrate.23 Subsequently, the Schimmel and co-workers also concluded that the His73 and Lys156 residues are well-positioned to partake in catalysis.25 Consequently, it was proposed that the His73 residue is neutral and acts as the required mechanistic base (Scheme 1). Specifically, it promotes activation of W1 through proton abstraction thus enabling the oxygen of W1 to nucleophilically attack the Ccarb center of the misacylated substrate Ser-tRNAThr. This ultimately results in cleavage of the labile

carbC—O3′

ester bond. The regenerated free

Ado763′O-tRNA

oxyanion is neutralized via a second proton transfer from the nearby Lys156 residue through a


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bridging water molecule (W2). The enzymatic use of a neutral histidyl as a mechanistically important base, such as in serine proteases and flavocytochrome b2, is widely documented.26,27

Scheme 1. Schematic illustration of the proposed post-transfer editing pathway as catalysed by bacterial E. coli ThrRS in which the mechanistic water nucleophile is activated by His73.

Recently, however, based on experimental oxidative stress studies on the editing domain of ThrRS, it has alternatively been suggested that the active site Cys182 residue may instead be the base, though acting indirectly via the

Ado762′OH

to activate the water.28,29 In particular, it was

observed that treatment of E. coli ThrRS by H2O2 resulted in formation of a Cys182-derived sulfenic acid (Cys182SOH). For such a species to be formed the Cys182 must be in its thiolate form. Notably, this oxidative modification significantly increased misincorporation mistakes, and it was concluded that this was due to inhibition of the post-transfer editing of Ser-tRNAThr by ThrRS. Furthermore, and in contrast to the previously proposed mechanism above, it was suggested that of the three active site histidyl residues (His73, His77, and His186), His73 and His186 are both protonated and help activate the thiol of Cys182 and stabilize its thiolate form.28 These studies29 also noted that the editing site of ThrRS is similar to the active site of cysteine proteases, which are known to form sulfenic acids, and that oxidation of cysteine residues to sulfenic acids often occurs in ROS signaling proteins.30 Moreover, it has been noted that the


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editing sites of AlaRS and ThrRS share considerable sequence similarity and that their active sites contain the same conserved residues; namely, two histidyl residues, and a cysteinyl.37 Indeed, in the editing site of AlaRS the cysteinyl (Cys666) is in a nearly identical orientation to that of Cys182 in the editing site of ThrRS. Furthermore, mutation of Cys666 to alanine severely inhibited the ability of the editing site of AlaRS to deacylate Ser-tRNAAla. Hence, it has been suggested that these two aaRSs may share a common post-transfer editing pathway.31-33 In the current study we have conducted a detailed, systematic multi-scale computational investigation using MD simulations and ONIOM(QM/MM) methodology to gain insight into the post-transfer editing mechanism in E. coli ThrRS. In particular, we have examined both the previously proposed hydrolytic editing mechanisms of ThrRS in which either His73 or Cys182 acts as the mechanistic base, as well as possible alternate mechanisms and for a range of different potential active site protonation states.

COMPUTATIONAL METHODS: Molecular Dynamics Simulations. The high-resolution experimental X-ray crystal structure of the editing domain of E. coli threonyl-tRNA synthetase with the substrate analogue seryl-3'aminoadenosine (SerAA) bound within the active site (PDB ID: 1TKY)24 was selected as the template for all chemical models used herein. The bound SerAA was mutated to the desired substrate (Ser-AMP) by replacing the relevant bridging –NH– with an oxygen atom. Hydrogen atoms were added as appropriate and according to protonation states as determined from pKa values calculated by both PROPKA 3.134 and the default tool in the Molecular Operating Environment software.35 Unconstrained Molecular Mechanics (MM) energy minimization using the AMBER12 forcefield was then performed on the generated model. The model was then solvated by adding a 6 Å layer of water around the entire complex (total 5649 water molecules for model A and 4467 for model B) followed by a second MM energy minimization which terminated once the root mean square gradient fell below 0.01 kJ/mol·Å2. The resulting complex


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was initially submitted for 100 ps equilibration from 0 to 300 K at constant pressure using the default settings of MOE which includes cut-offs for long range interactions of 10 Å. The mechanisms examined herein can be grouped into two broad categories; His73- or Cys182-promoted editing. Hence, two template enzyme-substrate complexes were generated. For the former scenarios, i.e. His73 acts as the base, the complex contained neutral His73, His186, and Cys182. These protonation states are consistent with their predicted pKa values (see above). For the alternate scenarios in which Cys182 acts as the base, consistent with the experimentally28 suggested ionization states the enzyme-substrate complex instead contained protonated His73 and His186 residues while Cys182 was deprotonated. Under constant pressure and temperature, the generated structures were then subjected to a second simulation for 500 ps, under the same conditions and 2 fs time steps, using the NAMD engine.36 The final conformation obtained in each 500 ps simulation was then checked to ensure it was a suitable representative structure, and then MM minimized as above. Suitable QM/MM chemical models were then generated by truncating the resulting complex to include all residues and waters within 10 Å of the substrate. QM/MM Investigations. A two-layer ONIOM(QM/MM)37,38 approach was used with the above derived chemical models being divided into QM- (high-level) and MM-subsystems (low-level) as shown in Figure 1. The substrate, mechanistically important residues, as well as select active site water molecules were included in the QM-region. The surrounding protein and remaining waters, were placed in the low-layer and described using an appropriate MM method. This methodology has previously been successfully applied to explore different catalytic mechanisms.39 All ONIOM(QM/MM) calculations were performed using the Gaussian09 software package.40 For those chemical models used to examine mechanisms in which His73 acts as the base the QM region contained: the substrate (Ser-Ado); the charge stabilizing residues Tyr104 and Gly95; the proposed catalytic residues His73 and Lys156; and Asp180 due to its role in orienting the substrate appropriately by formation of a salt bridge with the substrate’s α-NH2. In addition, in the appropriate MD simulations an active site water (W1) molecule was observed to be hydrogen


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bonded to the His73 residue and concomitantly in close proximity to the Ccarb of the substrate. Meanwhile, a second water (W2) molecule was hydrogen bonded to the side-chain amine of Lys156. Hence, both of these bridging water molecules (W1 and W2) were also included. The QM-region consisted of a total of 111 atoms and is shown in Figure 1a. For the chemical models used to examine mechanisms in which Cys182 acts as the base the QM-region was expanded to include the Cys182 and His186 residues. These QM-regions contained a total of 121 atoms and are shown in Figure 1b. Moreover, for all chemical models the α-carbon atoms of the amino acids in the low layer (at least one residue away from the QM layer) were held fixed at their initial positions in order to ensure the structural integrity of the model.

Figure 1. The template QM-region models used to study possible catalytic mechanisms of the ThrRS editing domain in which either (a) His73 or (b) Cys182 acts as the required base.

To obtain optimized structures, each QM layer was treated using the hybrid DFT method B3LYP,41-43 widely applied in the study of catalytic and enzymatic mechanisms,44 in conjunction with the 6-31G(d,p) basis set. To enhance modelling of non-covalent and dispersion interactions, the empirical D3 dispersion correction by Grimme was applied.45 This has previously been shown to significantly improve the accuracy of kinetic barriers.46 The AMBER96 force field was used for the MM region.47 Frequency calculations were also performed at this level of theory, ONIOM(B3LYP-D3/6-31G(d,p):AMBER96), to determine the nature of the stationary points and


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to calculate Gibbs free energy correction values (ΔGcorr). Relative energies were obtained via single point energy calculations on the above structures at the ONIOM(B3LYP-D3/6311+G(2df,p):AMBER96) level of theory with inclusion of the corresponding ΔGcorr. It is noted that all ONIOM calculations were performed within a mechanical embedding formalism.

RESULTS AND DISCUSSION: As noted above it has been suggested that either His73 or Cys182 acts as the base to facilitate and promote editing through activation of an active site water. However, this is complicated by the existence of several acid/base residues within the editing site. In addition, once a base has gained a proton it could, in some scenarios, potentially then act as an acid. Consequently, in this present study the mechanisms studied can be categorized as falling into either His73- or Cys182promoted editing mechanisms. However, for each of these we have examined several possible variations that differ by, for example, the protonation states of other editing site residues. In total, 11 different possible mechanisms have been elucidated and compared in terms of their chemistry and thermochemical feasibility.

I. His73-promoted Editing Mechanisms. (a) His73-promoted editing. The editing mechanism of archaeal ThrRS proceeds through a substrate-assisted mechanism in which the hydrolysis of the

carbC–O3′Ado76

Ado762′OH

group of the tRNAThr moiety facilitates

bond.23 In contrast, in the presently obtained optimized structure

of the reactive complex (IRC) of the editing site of bacterial ThrRS, shown in Figure 2, the substrate is positioned such that no hydrogen bonding interaction is observed between the substrate’s

Ado762′OH

group and the nucleophilic active site water (W1). Instead, W1 is in close

proximity to the catalytic His73 with which it forms a moderately strong hydrogen bond, r(His73N…HW1) = 1.88 Å. In addition, W1 is positioned near the substrate's Ccarb center at a distance of r(W1O…Ccarb) = 3.17 Å, Figure 2. Thus, it seems suitably positioned for proton transfer onto His73 and nucleophilic attack at Ccarb. Lys156, specifically its side chain


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ammonium, forms a strong hydrogen bond with a second water (W2), r(Lys156H…OW2) = 1.81 Å, which also positions it near to the substrate. Meanwhile, Asp180 helps hold the substrate in more catalytically productive conformation through formation of an Asp180COO−…H3N+SerAA salt bridge, r = 1.64 Å. The substrate’s Ocarb also forms a hydrogen bond with the backbone –N(H)– of Gly95 at a distance of 1.89 Å. Optimized structures of stationary points (energy minima and transition states) along the mechanism, together with key distances, are shown in Figure 2. The corresponding free energy surface (FES) is shown in Figure 3. The reaction is initiated by the His73 imidazole abstracting a proton from W1 while concomitantly, the water's OW1 nucleophilically attacks the Ccarb center of the substrate. This step proceeds via IaTS1 at a cost of 27.6 kcal/mol relative to IRC, suggesting an unfeasibly highenergy barrier. In IaTS1 the proton liberated from W1 has significantly shifted toward the His73 residue as indicated by the distances r(W1O…HW1) = 1.41 Å and r(W1H…N Furthermore, the oxygen of the developing

W1OH

–

I

Ia

= 1.16 Å.

moiety is now only 1.75 Å away from the

substrate’s Ccarb center having formed a weak partial bond. Collapse of energetically stable tetrahedral intermediate

H73)

Ia

TS1 generates the

IC1, lying 21.6 kcal/mol higher in energy than

RC, Figure 3. His73 is now protonated, r(H73N–H) = 1.06 Å, while the partial

has shortened markedly to 1.54 Å. Concomitantly,

__ carbC Ocarb

and

carbC–OW1

carbC–O3′Ado76

bond

lengthen by

0.06 and 0.14 Å, respectively, Figure 2. These reflect the change in hybridization of Ccarb toward pseudo sp3-hybridization and stabilization of the increased negative charge on Ocarb by the moderately strong hydrogen bond of length 1.76 Å it forms with the –N(H)– of Gly95. The next and final stage of the mechanism is cleavage of the formation of the

Ado76O3′H

carbC–O3′Ado76

bond and

group. Examination of IaIC1 suggests that there are potentially three

processes by which this could occur. Namely, it may involve proton transfer from the side chain ammonium of Lys156 through W2 directly onto Ado76O3′ or indirectly via Ocarb. Alternatively, the now protonated imidazole of His73 may act as an acid and directly protonate

Ado763′-oxygen.

Each mechanism was examined but only the two most feasible are shown in Figures 2 and 3.


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K156

K156

H

O

O

1.86

H O

H 3N

H

N

G95 O

K156

I94

O

Y104

H2 N

H

D180

Y104

G95

OH N

O

3.06

N

OH

H2 N

O

N

D180

O

H

O

O

N

NH 2

N

O

H O

G95

O H

OH O

H

O

O

H

H2 N

H

N

N H

N

N N

O

H

NH 2

N

O

O

HO

HO

1.70 H

N

N H

H73

IaTS1

OH

1.77

O

D180

HO

1.41

H

N H

O

N

N

1.75

I94

H N 1.16

1.88 H73

O H

1.75

HO

H O

IaRC

H

H

NH 2

H

Y104

H 3N

H

NH 2

N

O HO

N

O

H

O

I94

O

1.81

N H

H73

IaIC1

1) Y104

K156

2)

K156

Y104 O O

G95

H 2N

I94

O

NH 2

H

O

1.66 O

H

O

N

O

O

NH 2

O

O

H

Y104

O 95G

H

H

H

O

H

O

D180

H

1.81 H

O N N

1.64

HO

O

N

NH 2 N

D180

O

H

H2 N

H73

N H

O

Y104 O

N H

N N

NH 2 N

O

H

H73

HO

H

NH 2

N

O

O

N

N N

OH

H

H N H

OH

O

N

HisIC2 2

H O

D180

OH

2

1.51 1.37 O

1.11

H2 N

O

H

H

I94

N H

O H

N

O H

G95

OH O

O

HO

OH

H N

IaTS3 2

H O

H

H

O

H73

1.09 NH

H 1.51

N

O OH

1.25

OH

NH 2 N

K156

NH 2

O I94 G95

O

O

NH 2

Y104

H

1.30 H

O

N

K156

NH 2

I94

N

N

2.16 H N

IaTS2 1

K156

O

N

O

1.33

N N H

H73

IaPC

OH

H

O

HO

OH

H

NH 2

H O 1.66

1.12

H

N

N

H

H

N

D180

NH 2

N

I94

H

O

OH

2.81 O

HO

G95

H

H

H

H

D180

O

1.67

N

H O

O

N

HisTS2 2

H73

N H

Figure 2. Schematic illustration of the optimized structures obtained (see Computational Methods), select distances in Angstroms, for the His73 promoted mechanisms obtained with Lys156 protonating either the (1) Ado763′O or (2) Ocarb centers of the Ser-tRNAThr substrate.


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Figure 3. Free energy surfaces (kcal/mol) calculated (see Computational Methods) for the ThrRS editing domain mechanisms in which His73 acts as a base with Lys156 acting as an acid and protonating either the Ocarb center (blue line) or Ado763′-O (red line).

1.

Direct water mediated protonation of

Ado763′O

by Lys156. The side chain ammonium of

Lys156 is able to protonate the 3′OAdo76 center of IaIC1 via a mediating water W2. This proceeds through

Ia

TS21 with a free energy barrier of 33.4 kcal/mol, Figure 3. In this mechanism the

proton transfer represents the rate-limiting step. In addition, the high barrier suggests that this mechanism is unlikely to be enzymatically feasible. It is noted that

Ia

TS21 appears to be a

dissociative transition state since the proton has essentially transferred onto the

Ado763′-oxygen

while the Ado763′O…Ccarb bond has concomitantly significantly elongated to 2.16 Å, Figure 2. The resultant product complex IPC lies 4.3 kcal/mol higher in energy than IRC; that is, this mechanism is also thermodynamically unfavorable. In IPC the Lys156 side chain amine is now neutral with the Ado763′OH group having been fully formed, Figure 2.


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2.

Indirect water-mediated protonation of Ado763′O by Lys156. Lys156 may first transfer its

ammonium proton via W2 onto the oxyanionic Ocarb center in intramolecular proton transfer can occur from

carbOH

Ia

IC1. Subsequently, an

onto 3′OAdo76 of the leaving tRNA moiety,

Figure 2. The first step in this process proceeds through IaTS22 at a cost of 32.6 kcal/mol relative to IRC, Figure 3. While IaTS22 is 0.8 kcal/mol lower in energy than IaTS21, it is still too high to be enzymatically feasible. Furthermore, W2 has almost wholly transferred its proton onto the substrates Ocarb center as indicated by the distances r(W2H···OW2) = 1.37 Å and r(W2H···Ocarb) = 1.11 Å, Figure 2. The resultant tetrahedral 1,1-diol intermediate complex (IaIC2) is 1.1 kcal/mol higher in energy than IaIC, and 22.7 kcal/mol higher in energy relative to IRC, Figure 3. The subsequent proton transfer from the newly formed -carbOH group onto the Ado763′-oxygen proceeds via the 4-membered-ring ITS3, Figure 2. As noted earlier,

Ado762′OH

in the optimized

model herein is oriented such that it is unable to readily participate in this step to help form a 6membered-ring transition state. The energy required for this second step is extremely high with a barrier of 69.6 kcal/mol relative to IRC, Figure 3, clearly indicating that editing using this possible mechanism is enzymatically unfeasible. It is noted that even if an alternative pathway for this second proton transfer reaction was possible, the barrier for proton transfer through IaTS22 in itself is higher than likely to be enzymatically feasible. The final product complex formed, IPC, is the same as that obtained for the above described direct water meditated protonation mechanism. Hence, this possible alternate mechanism is also thermodynamically unfavorable. 3.

Protonation of

Ado763′O

by His73-H+. His73 has also been proposed to act as an

acid/base catalyst and thereby the proton transferred to the

Ado763′-oxygen,

cleaving the

carbC—

O3′Ado76 bond, may originate from a protonated His73 formed in the first step of the mechanism, Figure 2.25 However, the relative energy of the required transition state is 45.8 kcal/mol while . the corresponding product complex is 24.1 kcal/mol higher in energy than IRC.

(b) His73-promoted editing through self-cyclization/lactone formation: In the reactive complex IRC it was observed that the substrate's serinyl side-chain, and in particular its b-OH


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group, could potentially undergo a conformational change. Specifically, it could rotate such that it no longer interacts with the side-chain carboxylate of Asp180 as is common,5,22 but instead could potentially interact with His73. Such a geometry and interaction may facilitate an alternate editing pathway in which cleavage of the aminoacyl-tRNA ester bond proceeds through a 4membered lactone ring, Figure 4. Similar cyclizations have been suggested to play a role in editing of other misacylated substrate's including the pre-transfer editing mechanism of MetRS previously reported by our group.20

Figure 4. Schematic representation of the optimized complexes, with select distances shown in Ångstroms, obtained for a His73-promoted self-cyclization editing mechanism. The relative free energies (kcal/mol) are also given in brackets.


14

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ACS Catalysis

Rotation around the substrate's serinyl side chain C__C bond leads to formation of the intermediate

Ib

IC1 5.3 kcal/mol higher in energy than IRC. It is noted that the scan was

performed as the rotational barrier was quite small. Indeed, after empirical free energy corrections were included the barrier was estimated to be slightly lower than 0.0 kcal/mol, reinforcing that in vivo this rotation can occur essentially without a barrier. In

Ib

IC1 the

substrate’s serinyl b-OH is strongly hydrogen bonded to the neutral imidazole of His73 whilst also simultaneously in close proximity to the substrate's Ccarb center with distances of r(H73N···HOH) = 1.76 Å and r(OHO…Ccarb) = 2.79 Å. The b-OH oxygen is then able to nucleophilically attack the Ccarb center with concomitant transfer of the hydroxyl's proton onto His73. This step proceeds through IbTS2 at a quite high cost of 31.4 kcal/mol relative to IRC. This may again be due in part to the increasing anionic charge on Ocarb during this step being stabilized by only a single hydrogen bond with the -NH- of Gly95. The resulting tetrahedral intermediate IbIC2 lies slightly lower in free energy than IRC by −3.4 kcal/mol. Importantly, it should be noted that the intermediate ligand has shifted position slightly so that the Ocarb oxyanion center is now hydrogen bonded to the protonated imidazole of His73 (i.e. His73-H+). This suggests that the latter residue may be able to transfer its proton onto the Ocarb oxyanion center in order to generate a neutral lactol. And indeed, such a proton transfer can occur essentially without a barrier via IbTS3, as indicated by the fact that its calculated free energy after empirical corrections is lower than that of

Ib

IC2. Importantly, this results in

formation of the low energy lactol IbIC3 which lies markedly lower in energy than IRC by 42.5 kcal/mol and is in fact the lowest energy complex along this pathway. The lowest energy pathway for the subsequent cleavage of the

carbC−3′OAdo76

an intramolecular proton transfer from the newly formed -carbOH group onto the This occurs via the four-membered ring transition state

Ib

bond involves Ado763′-oxygen.

TS4 with a barrier of 33.3 kcal/mol

relative to IbIC3. This is the rate-limiting step of the reaction but its value suggests that at least in the editing domain of ThrRS this pathway is enzymatically unfeasible. The product formed, IbPC,


15

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Page 16 of 34

while thermodynamically favoured compared to IRC by 13.3 kcal/mol, is much higher in energy than IbIC3 by 29.2 kcal/mol, Figure 4.

II. Cys182-promoted editing Mechanisms. As noted above, it has also been suggested that the active site cysteinyl Cys182 may instead be able to act as a base to promote/catalyse the editing mechanism. Thus we systematically examined possible mechanisms through which this may occur, with a key difference between them being the protonation states of the conserved active site His73, Cys182 and His186 residues. However, as for the above mechanisms, in some cases several scenarios were considered based on the source of the proton in the second stage and/or the site of substrate to which it is initially transferred. It is noted that as for the above His73-promoted editing mechanisms, the side chain of Lys156 was considered to be in its protonated form.

(a) Deprotonated Cys182 with protonated His73 and His186 (Cys182S–/His73-H+/His186-H+). In the QM/MM optimized structure (see Computational Methods) of the reactant complex

IIa

RC,

the anionic thiolate is stabilized by hydrogen bonds with the protonated imidazoles of His73 and His186 with distances of r(HisNH···SCys) = 2.26 Å and 1.90 Å, respectively, Figure 5. In addition, it forms a moderately strong hydrogen bond with the nucleophilic water W1 with r(W1H…SCys182) = 1.89 Å. Similar to that observed in

Ia

RC the Asp180 anchors the substrate via salt bridge

formation with the substrate’s α-NH3+ group at a distance of 1.51 Å. Meanwhile, the W2 molecule is positioned close to the other side of the substrate through hydrogen bonding to the backbone carbonyl oxygen of the nearby Ile94 residue with r(W2H…OIle94) = 1.65 Å, Figure 5. Deprotonation of W1 by the Cys182 thiolate facilitates nucleophilic attack of the oxygen of W1 on the substrate’s Ccarb center. This reaction proceeds through the transition state

IIa

TS1 at a

cost of 17.4 kcal/mol relative to IIaRC, which is 10.2 kcal/mol less than the corresponding step in the above described His73-promoted mechanisms, Figures 3 and 6. It is noted that it represents a late transition state as the W1 proton is substantially shifted toward the sulfur atom and the W1


16

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ACS Catalysis

oxygen has moved quite close to the Ccarb as indicated by their distances of r(W1H···SCys182) = 1.49 Å and r(W1O···Ccarb) = 1.73 Å, Figure 5. Similar to ITS1 in the His73-promoted mechanisms, the substrate’s Ocarb center is stabilized by formation of a strong hydrogen bond with the backbone of Gly95, r(Gly95N(H)…Ocarb) = 1.78 Å which, due to the increased negative charge on Ocarb, is 0.26 Å shorter than the same interaction in

IIa

RC, Figure 5. In addition,

however, it also forms a strong hydrogen bond with a nearby active site water, r(carbO…HW) = 1.81 Å, which is simultaneously hydrogen bonded to the protonated imidazole of His73. The latter is made possible due to the Cys182 now being neutral and remains hydrogen bonded to His186-H+. This additional stabilizing hydrogen bond of Ocarb may help explain the markedly lower reaction barrier obtained for this step. Collapse of

IIa

TS1 in leads to the formation of

intermediate complex IIaIC1, which possesses typical carbC−OH and RS−H bonds (r = 1.49 Å and 1.36 Å, respectively). Notably,

IIa

IC1 lies 13.7 kcal/mol higher in energy than

IIa

RC, which is

markedly lower than the relative energy (21.6 kcal/mol) than the corresponding intermediate IIC1 in the His73-promoted mechanisms, Figure 3. As for the above mechanisms in which His73 acted as the base, there are three possible proton donors (Lys156, His73, and Cys182) present for the continuation of the mechanism and cleavage of the

Ado763′O—Ccarb

bond. Each of these possibilities was investigated; only the most feasible

mechanism obtained for each is discussed herein. 1. Direct protonation of

Ado763′O

by Lys156. In

IIa

IC1

Ado763′O

is strongly hydrogen bonded

to a proton of W2 at a distance of 1.79 Å. Notably, W2 is simultaneously also positioned near the side chain ammonium of Lys156. For the above His73 catalyzed process, proton transfer from Lys156 onto the Ado763′-oxygen gave the lowest barriers. For this present scenario proton transfer from Lys156 onto

Ado763′O

can occur with the involvement of the bridging water molecule, but

with a high free energy barrier, via

IIa

TS21 of 32.5 kcal/mol relative to

IIa

RC (Figure 6). This

step also appears to share structural similarities to that described above in the analogous His73promoted mechanism that proceeds via IaTS21. Namely, IIaTS21 also appears to be a dissociative transition state as the H···N+Lys156 distance has elongated significantly to 1.85 Å (i.e. has been


17

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transferred onto W2) while the

carbC−O3′Ado76

Page 18 of 34

bond has also been essentially wholly cleaved as

indicated by its distance of 1.71 Å, Figure 5. Collapse of

IIa

TS21 leads to formation of the

product IIaPC1 in which the tRNA's Ado76 ribose sugar and the noncognate serine amino acid are separated by a distance of 2.84 Å, Figure 5. Notably, this overall pathway was found to be thermodynamically favorable as IIaPC1 is 33.6 kcal/mol lower in energy than IIaRC, Figure 6.

Figure 5. Schematic illustration of the optimized structures obtained, with select bond distances shown in Ångstroms, for the mechanism in which Cys182 acts as a base to activate an active water, and with Lys156 acting as acid and directly protonating the Ado763′-oxygen.

2. Protonation of

Ado763′O

involving His73-H+. In

IIa

IC it was observed that with

neutralization of the Cys182 thiolate, the protonated imidazole of His73 (His73-H+) has switched position and is hydrogen bonded to a third editing site water molecule (W3). Thus, His73-H+ may in fact be able to act as an acid and transfer its proton on the intermediate. It was found that in


18

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ACS Catalysis

fact His73-H+ is able to effectively transfer its proton via W3 onto the oxyanionic Ocarb center of IIa

IC. Furthermore, this effectively occurs without a barrier via

slightly lower free energy relative to IIa

IIa

IIa

TS22 as suggested by its

IC (Figure 6). This low barrier also reflects the fact that

TS22 occurs early along this step; the His73N—H distance has lengthened only slightly to 1.15 Å

and the distance to the OW3 center is still quite long at 1.36 Å. meanwhile, the W3O…H distance for the proton W3 concurrently donates to the substrate’s Ocarb center has increased significantly to 1.21 Å and the corresponding WH…Ocarb distance is relatively short at 1.25 Å, Figure 7.

Figure 6. FES obtained (kcal/mol) for the three possible editing mechanisms elucidated in which Cys182 acts as a base and His73 and His186 are protonated and the required acid for cleavage of the Ado763′O—Ccarb bond is: Lys156 (black line and labels); His73 (red line and labels); or Cys182 (blue line and labels).


19

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Page 20 of 34

Indeed, the subsequently formed 1,1 diol intermediate IIIC22, containing a tetrahedral Ccarb center, lies 11.0 kcal/mol lower in energy than the initial complex IIaRC, Figure 6. The next and final step is transfer of the proton from the newly formed Ccarb–OH groups onto the 3′OAdo76 center. The lowest energy pathway for such a transfer was found to occur involve the –OH group formed from W1 and proceeds via the 6-membered ring transition state

IIa

TS33. However,

energetically this step has a high energy barrier of 33.3 kcal/mol with respect to

IIa

IC2 and is in

fact the overall rate-limiting step for this possible pathway. This is likely at least partly a reflection of the high pKa (i.e. low acidity) of the Ado762′OH group which is integrally involved in this step (Figure 7). However, the resulting product complex (IIaPC2) formed, in which the Ado763′O—Ccarb

bond is fully broken, is thermodynamically very favorable having an energy

relative to IIaRC of −50.0 kcal/mol (Figure 6).

Figure 7. Schematic illustration of the optimized structures, with selected bond lengths shown in Ångstroms, obtained for the IIa editing mechanism (i.e., initial active site contains Cys182S– /His73-H+/His186-H+) in which cleavage of the

Ado763′O—Ccarb

bond involves proton transfer

from His73 (2: red labels) or Cys182 (3: blue labels).


20

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ACS Catalysis

3.

Protonation of

Ado763′O

involving

Cys182SH.

Alternatively, the neutral thiol of Cys182

formed during the first step may act as an acid to facilitate cleavage of the Ado763′O—Ccarb bond, Figure 7. More specifically,

Cys182SH

could transfer its proton onto the nearby

Ado762′OH

moiety

while the latter concomitantly transfers its proton onto the Ado763′-oxygen breaking the Ado763′O— Ccarb bond. However, this proceeds via IIaTS23 with an energy barrier of 27.9 kcal/mol relative to IIa

RC (14.2 kcal/mol relative to

IIa

IC1) and is the overall rate-limiting step for this pathway.

Though it is high, it is the lowest barrier amongst the three possibilities elucidated for when the editing site initially contains a deprotonated Cys182, and protonated His73 and His186, Figure 6. The resulting product

IIa

PC3 is the thermodynamically most favored of these three related

pathways with an energy 51.0 kcal/mol lower than that of IIaRC.

(b) Deprotonated Cys182 with neutral His73 and His186 (Cys182S–/His73/His186). It is noted that all related AlaXps enzymes there are three residues (His9, His13 and Cys116) that are positioned similarly to those of His73, His186 and Cys182 in the editing site of ThrRS.7,48 In addition, the Zn(II) binding-site in the aminoacylation site of ThrRS contains a similarly arranged deprotonated cysteinyl and two neutral histidyl residues.15 Furthermore, the pKas predicted using PROPKA for these three residues in the editing site of ThrRS suggested that Cys182 may be deprotonated with His73 and His186 being neutral. Hence, we considered mechanisms in which these where their initial protonation states in the editing site with the optimized structures and thermochemistry of the two possible pathways elucidated given in Figures 8 and 9. We also performed an in silico His73Ala mutation of the enzyme and the pKas of

Cys182SH

in the native

and mutant enzymes were compared (not shown). An increase of ~1 unit was observed indicating that His73 may help ensure the Cys182 thiol is more likely to be deprotonated. In the QM/MM optimized structure of the corresponding reactive complex IIbRC, the thiolate of Cys182 forms a hydrogen bond with an active site water molecule (W1) with r(Cys182S…HW1) = 2.18 Å (Figure 8). Notably, this orients W1 such that its oxygen (OW1) is positioned just 2.64 Å from the substrate's Ccarb center, a distance which is significantly shorter by 1.40 Å than observed


21

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Page 22 of 34

in IIaRC. This suggests that W1 is well placed for the subsequent required nucleophilic addition, Figure 8. This seemingly better positioning may also be due to the fact that unlike in

IIa

RC, the

thiolate of Cys182 does not form a hdyrogen bonding network with His73 and His186 due to their being neutral. Instead, and in addition to its hydrogen bond with W1,

Cys182S

–

also forms a

moderately strong hydrogen bond of length 2.16 Å directly with the Ado762′OH group (Figure 8). This appears to help bind and orient the substrate in a more conducive conformation for subsequent reaction.24

1.70

1.73

1.67

1.78

2.64

2.00 1.30

2.18

2.16

2.28

1.59

IIb

IIbRC

III

IC

TS1

2)

1) K156 O G95

N

1.80

H H

O

1.55

H 2N

I94

O H

1.18

OH

1.73

O H

NH 2

D180

O

NH 2

N O

H

HO

O H

1.24

N

N

O

N OH

HS

C182

IIb

PC

IIb

TS2

IIb

TS22

2.83 1.04

1.82 2.14

1.55 1.49 1.46

IIb

PC2

Figure 8. Schematic illustration of the optimized structures (select bond lengths shown in Ångstroms) obtained for the editing pathways in which Cys182 acts as the base in the presence of


22

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ACS Catalysis

neutral His73 and His186, and where either Lys156 (1: black labels) or Cys182 (2: red labels) acts as the required acid for the second proton transfer.

The analogous pathway for activation and nucleophilic attack of W1 on the substrate, as obtained for

IIa

RC was elucidated. Again, the Cys182 thiolate can abstract a proton from W1

while the latter's oxygen (OW1) nucleophilically attacks the substrate's Ccarb center, Figure 8. In contrast, however, this reaction proceeds via lower than for the analogous reaction via

IIa

IIb

TS1 at a cost of just 9.9 kcal/mol, 8 kcal/mol

TS1. (Figure 9). Indeed, of all systems considered

herein, this is the lowest barrier for activation and subsequent nucleophilic attack by the active site water. It is noted that in IIbTS1, the Cys182S…HO2′Ado76 interaction has been maintained though it has lengthened slightly to 2.28 Å. Meanwhile, the developing negative charge on Ocarb is again stabilized by a strong hydrogen bond with the Gly95N−H group; r(Gly95N(H)…Ocarb) = 1.67 Å. This latter distance is 0.12 Å shorter than observed in the IIaTS1. As before, this results in formation of an oxyanionic tetrahedral intermediate (IIbIC), in which r(Gly95N(H)…Ocarb) has shortened markedly to 1.58 Å (Figure 8). Significantly, as shown in Figure 9, higher in energy than the initial reactive complex

IIb

IIb

IC is just 4.4 kcal/mol

RC. Hence, in terms of relative free energy,

it is the lowest in energy of all such intermediates IIC, IIaIC, and IIbIC obtained herein. As for the other systems examined, subsequent breaking of the Ado763′O—Ccarb bond requires an acid to transfer its proton, directly or indirectly, onto the

Ado763′-oxygen.

In this present

scenario, the most probable proton sources are the ammonium of Lys156 or the now neutral thiol of Cys182. Hence, both possible pathways were considered. 1.

Protonation of

Ado763′O

involving Lys156. In both

IIb

RC and, in particular,

second active water (W2) is positioned near both the ammonium of Lys156 and the oxygen. Indeed, in

IIb

IIb

IC, a

Ado763′-

IC W2 simultaneously forms strong hydrogens bonds with both of these

groups; r(Ado763′O…HOW2) = 1.78 Å and r(Lys156NH…OW2) = 1.70 Å, Figure 8. Water-mediated transfer of a proton from the ammonium of Lys156 onto the through

IIb

Ado763′-oxygen

is able to proceed

TS21 with an enzymatically feasible barrier of 20.8 kcal/mol relative to the reactive


23

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Page 24 of 34

complex IIbRC, Figure 9. As for all other mechanisms elucidated this barrier is the rate-limiting step of the overall mechanism. Importantly, however, it is the lowest barrier obtained for this step of all systems considered herein. In the product,

IIb

PC1, the noncognate serine and tRNAThr

formed are now significantly separated as illustrated by r(carbC…O3′Ado76) = 3.19 Å. Notably this product complex was also thermodynamically the most favorable of all obtained being 53.6 kcal/mol lower in energy relative to

IIb

RC, Figure 9. Collectively, the free energy values

obtained for this pathway, both kinetically and thermodynamically, support the possibility of the editing mechanism at least involving Cys182 as the water activating base.

Figure 9. FES obtained (kcal/mol) for the two possible editing mechanisms elucidated in which Cys182 acts as a base and His73 and His186 are neutral and the required acid for cleavage of the Ado763′O—Ccarb

bond is: Lys156 (blue line and labels); or Cys182 (red line and labels).


24

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ACS Catalysis

2.

Protonation of

of Cys182 to the

Ado763′O

Ado762′OH

involving Cys182. The close proximity of the now neutral thiol

group presents the possibility that Cys182 may be able to act as the

acid with the assistance of the

Ado762′OH

group to protonate the

Ado763′O

center, Figure 8.

However, this occurs via IIbTS22 at a cost of 26.7 kcal/mol relative to IIbRC, which is higher than obtained above for Lys156 acting as the acid. Furthermore, the resulting product energetically less favoured than

IIb

PC1 relative to

IIb

IIb

PC2 is

RC. Thus, Lys156 appears to be preferred

kinetically and thermodynamically as the required acid.

(c) Activation of Cys182 by His73, in the presence of a protonated or neutral His186. It has been suggested that one role His73 may be to activate the Cys182 thiol.28 In this scenario both Cys182 and His73 would necessarily initially be neutral, while His186 may be either protonated or neutral. For completeness, both possible scenarios were examined; that is, we examined possible mechanisms which being with activation of Cys182. For the case in which initially His73 and Cys182 are neutral but His186 is protonated, the pre-reactive complex lies 10.3 kcal/mol lower in energy than the reactive complex formed after activation,

IIa

RC. Furthermore, proton transfer

from the thiol of Cys182 onto His73 occurs with a barrier of 21.6 kcal/mol. However, while this barrier is feasible, the subsequent editing mechanisms that proceed via

IIa

RC all have barriers

that are likely enzymatically unfeasible (see Figure 6). Hence, this proposal was thus discounted. Alternatively, His73 may activate Cys182 in the presence of a neutral His186, i.e. all three conserved residues (His73, His186 and Cys182) are initially neutral. The free energy surface obtained for the resulting mechanism, and the corresponding QM/MM optimized structures, are shown in Figure 10. As before, the first step is deprotonation of the Cys182 thiol by His73;28 that is, the conversion of the pre-reactive complex This proton transfer occurs via

IIc

IIc

PRC into the required reactive complex

IIc

RC.

TS1 with a barrier of 21.7 kcal/mol. Notably, this is only 0.1

kcal/mol higher than obtained for the analogous step in the presence of protonated His186 suggesting that it does significantly influence the activation step. In the reactive complex


IIc

RC 25

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Page 26 of 34

Cys182 is now deprotonated and His73 protonated while His186 remains neutral. Importantly, it lies just 0.4 kcal/mol higher in energy than the pre-reactive complex IIcPRC, Figure 10.

W1

1.62

1.97

1.73

W1

H73 H73

K156 1.77

2.04

1.91

1.64 2.84

C183

C183

C183 C183 IIc TS2 IIc TS3

27.9 IIc TS1

25.1

21.7

IIc IC

16.7

IIc RC

IIc PRC

0.4

0.0

K156

W1

H73

2.16 1.29

1.59 1.05 W2 1.27

1.89 1.27 1.56

1.68

1.72

IIc PC

-43.2

1.16

C183

C183

Figure 10. FES obtained (kcal/mol), together with optimized structures with select bond distances shown (Angstroms), for the ThrRS editing domain's mechanism IIc in which Cys182 acts as a base, but Cys182, His73 and His186 are all initially neutral.

In IIcRC the Cys182 thiolate is hydrogen bonded to W1 with distance r(Cys182S…HW1) = 2.04 Å. As observed in the other mechanisms examined herein, W1 is positioned in proximity to the substrate's Ccarb center with a

W1O

…

Ccarb distance of 3.22 Å. The thiolate of Cys182 again

facilitates nucleophilic attack of W1 at the substrate's Ccarb center by abstracting one of its protons. In this present system this step proceeds through

IIc

TS2 at a cost of 27.9 kcal/mol

(Figure 10). This is the highest barrier obtained of all mechanisms considered herein for the formation of a tetrahedral intermediate complex. In fact, it is 18.0 kcal/mol higher than that obtained for the corresponding step (i.e.

IIb

TS1, Figure 9) in the mechanism found to be

preferred of all those considered herein (IIb1). The oxyanionic tetrahedral intermediate formed,


26

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IIc

ACS Catalysis

IC, is 16.7 kcal/mol higher in energy than the initial pre-reactive complex

and final step is

Ado763′O—Ccarb

IIc

PRC. The next

bond cleavage. This again preferentially occurs via a water-

mediated proton transfer from Lys156 onto the requires just 8.4 kcal/mol with respect to

IIc

Ado763′-oxygen.

This proceeds via

IC; 25.1 kcal/mol relative to

IIc

IIc

TS3 and

PRC. In the product

complex IIcPC the now hydrolysed non-cognate serine and tRNAThr are 2.84 Å apart. In addition, its formation is thermodynamically favoured having a relative energy of −43.2 kcal/mol. However, while this is an exergonic mechanism, and more favourable than some of the alternative pathways considered, its barrier for the rate-limiting step in this case is thermodynamically less favorable than that of the preferred mechanism, IIb1 (see Figure 9).

CONCLUSION: In this study we have performed an extensive systematic MD and QM/MM investigation on the post-transfer editing mechanism catalyzed by the editing domain of ThrRS from Escherichia coli for deaminoacylation of mischarged Ser-tRNAThr. In particular, a range of possible scenarios have been examined in which either of the potential mechanistic bases His73 or Cys182 deprotonate an active site water to facilitate the latter’s nucleophilic attack on the substrate’s carbonyl center in a hydrolytic editing process. In addition, we examined the potential of a His73promoted pathway involving a non-hydrolytic self-cyclization/lactone formation. In total, the chemistry and feasibility of 11 different possible mechanisms, with different possible active site protonation states, were examined and compared. When His73 acts as the initial base five different mechanisms were considered in which it deprotonates either a nucleophilic water molecule (W1, resulting in hydrolytic editing) or the Ser-AMP substrate’s serinyl β-OH, resulting in lactone formation. For those pathways in which His73 activates W1 the mechanism essentially occurred in two stages: (i) nucleophilic attack of W1 at the Ser-AMP's Ccarb center to form a tetrahedral intermediate; and (ii) cleavage of the Ado763'O—Ccarb

bond with protonation of the Ado763'-oxygen. The first stage is a common one-step

reaction with a free energy barrier of 27.6 kcal/mol. For the second stage, several possible proton


27

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Page 28 of 34

sources were considered. The most favorable pathway proceeded via a one-step reaction in which Lys156 protonates the

Ado763'-oxygen

through a second active site water molecule (W2) with a

rate-limiting barrier of 33.4 kcal/mol. For the His73-promoted lactone formation pathway, deprotonation of the β-OH group with formation of the cyclic tetrahedral intermediate occurred with a barrier of 31.4 kcal/mol. The rate-limiting step of this pathway, however, was cleavage of the Ado763'O—Ccarb bond at a cost of 32.8 kcal/mol. For the Cys182-base scenarios, three chemical models with varying combinations of protonation states for the conserved His73, His186 and Cys182 residues were used to investigate the feasibility of six possible mechanisms. As with the alternate His73-promoted hydrolytic editing mechanism, these occurred in two stages as above. Of all mechanisms considered herein, the most kinetically and thermodynamically feasible pathway was obtained when both His73 and His186 are in their neutral states, and Cys182 is in its thiolate form (Figure 8; pathway 1). The first stage of the mechanism, Cys182 activation of W1 and formation of the tetrahedral intermediate, occurs in one step with a barrier of 9.9 kcal/mol. For the second stage, cleavage of the

Ado763'O—Ccarb

protonating the

bond, the most favorable pathway also occurs in one step with Lys156

Ado763'-oxygen

via W2 with a free energy barrier of 20.8 kcal/mol. The resulting

product complex, with a relative free energy of −53.6 kcal/mol with respect to its corresponding initial reactive complex, was also the most favored of all of the mechanisms. Hence, the present results support the conserved Cys182 residue as being in its thiolate form and acting as the required mechanistic base that initiates editing within the editing domain of E. coli ThRS. In several models a hydrogen bond was seen between the substrate’s serinyl β-OH and neutral His73 imidazole. This may suggest that the observed experimental importance of His73 is a result of a role in binding the substrate. Furthermore, it was observed to have a significant effect upon the acidity (and hence protonation state) of the Cys182 thiol. The histidine may thus also be important for providing an environment conducive to Cys182 acting as the mechanistic base. Importantly, given the noted similarities to the editing site of AlaRS and related enzymes that share the same catalytic motif, this mechanism may be more generally applicable.


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ACS Catalysis

Corresponding Author *Author to whom correspondence should be addressed; E-mail: [email protected]; Tel.: +1519-253-3000 Ext. 3992; Fax: +1-519-973-7098.

Author Contribution: The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Resources: We thank the Natural Science and Engineering Research Council of Canada (NSERC) for financial support. M.A. would also like to thank the Egyptian Cultural Affairs & Mission Sector for support at the beginning of his study. We thank SHARCNET for additional computational resources

ACKNOWLEDGMENT: We thank compute Canada for computational resources.

ABBREVIATION: aaRS, aminoacyl-tRNA synthetases; AlaRS, alanyl-tRNA synthetases; ThrRS, threonyl-tRNA synthetases; DFT, Density functional Theory; QM/MM, Quantum Mechanics/Molecular Mechanics; MD, Molecular Dynamics.

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Table of Contents Graphic.

Ser-tRNAThr

His73

? Cys183

Ser

tRNAThr


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A Molecular Dynamics - American Chemical Society

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